Method of analyzing coenzyme q-10 and two-electron reduction product thereof and analysis system

ABSTRACT

The present invention relates to an analysis method and analysis system that can accurately analyze the contents of coenzyme Q-10 and a 2-electron reduced form thereof in a specimen, in which human blood plasma as the specimen is mixed with isopropyl alcohol as a pretreatment and the coenzyme Q-10 and the 2-electron reduced form thereof are extracted to the isopropyl alcohol. Extracted liquid is stored at a temperature of 4° C. until the analysis is performed. The extracted liquid as an analytical sample is analyzed by an analysis system provided with a liquid-sending mechanism, a switching mechanism, a concentration column, a separation column, a reduction column, an ultraviolet absorption detector, and an electrochemical detector.

TECHNICAL FIELD

The present invention relates to an analysis method and analysis systemfor coenzyme Q-10 and a 2-electron reduced form thereof.

BACKGROUND ART

Coenzyme Q (Coenzyme Q: CoQ) is a benzoquinone derivative and is namedas a ubiquinone since it widely exists in the living world. Ahydroquinone obtained by 1-electron-reducing a ubiquinone is aubiquinol.

The compound name of the ubiquinone is2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone, and a great numberof homologs with n=1 through 12 isoprene units naturally exist, whereinn=10 for higher animals such as humans. In the following description, aubiquinone of humans, etc., is represented as coenzyme Q-10 and aubiquinol of humans, etc., is represented as a 2-electron reduced formof coenzyme Q-10, as long as no particular reference is made.

The 2-electron reduced form of coenzyme Q-10 has a strong oxidativeeffect and is considered to have an antiaging effect such as theprevention of cellular damage caused by active oxygen.

Oxidation stress is considered to be an undesired state for an organismin which the balance between oxidation and antioxidation in the organismis disrupted so as to shift toward oxidation, and it is considered thatthe ratio of the coenzyme Q-10 and the 2-electron reduced form thereofcan be a good marker for oxidation stress since it is considered toreflect the degree of oxidation stress.

Thus, since it is very useful to know the behaviors of the coenzyme Q-10and the 2-electron reduced form thereof, a method for accuratelyanalyzing these components is desired.

As a conventional analysis method, an ultraviolet absorption method,etc., is provided but it is easily subjected to the influence of a thirdsubstance and, therefore, a complicated pretreatment is required.

In recent years, a high-performance liquid chromatography (referred toas a HPLC below) has been widely used as a method that can attain ahighly sensitive and accurate analysis. For the detection of coenzymeQ-10, ultraviolet absorption at 275 nm is utilized. However, thesensitivity of the conventional analysis method by means of a HPLC isnot enough to detect coenzyme Q-10 in blood plasma.

For this reason, a method of reducing coenzyme Q-10 to a 2-electronreduced form thereof for quantifying the difference between thequantities of the coenzyme Q-10 before and after the reduction is alsosuggested. However, in this analysis method, it is necessary to performa pretreatment on a specimen and to twice perform sample injections intoa HPLC.

Therefore, the applicants previously suggested a method such that aftercoenzyme Q-10 and a 2-electron reduced form thereof are separated by areversed phase separation column 1 (LC-8 produced by SUPELCO), thecoenzyme Q-10 is reduced to a 2-electron reduced form thereof using areduction column 2 (SHISEIDO CQ produced by Shiseido Co., Ltd.) orcoulometric electrodes, which reduced forms are measured by anelectrochemical detector 3, as shown in FIG. 1 (For example, see SatosiYamasita and Yorihiro Yamamoto, ANALYTICAL BIOCHEMISTRY, 250, 66-73(1997)). Herein, in FIG. 1, reference numerals 4, 5, 6, 7, and 8 denotea mobile phase, a pump, a sample injector, a protection column, and anultraviolet absorption detector, respectively.

The highly sensitive simultaneous measurement of the coenzyme Q-10 andthe 2-electron reduced form thereof can be attained using the analysismethod described above. One example of an obtained chromatogram is shownin FIG. 2. In FIG. 2, “1” denotes a peak for the coenzyme Q-10 and “2”denotes a peak for the 2-electron reduced form of the coenzyme Q-10.

In this case, since the measurement is influenced by a water-solubleantioxidation substance in the specimen, such as vitamin C and uricacid, etc., a pretreatment is performed for subjecting the specimen toan extraction treatment with methanol/hexane so as to distribute thewater-soluble antioxidation substance into a methanol phase and thecoenzyme Q-10, etc., into a hexane phase.

However, when pretreatment is performed subjecting the specimen to theextraction treatment with methanol/hexane, the coenzyme Q-10 in thehexane extraction liquid is chemically unstable. After the pretreatmentand until the pretreated liquid is injected into a HPLC for analysis,the 2-electron reduced form of coenzyme Q-10 is oxidized at asubstantial rate as shown in FIG. 3. It is therefore necessary toperform analysis immediately after the extraction of the specimen. Forperforming an accurate analysis, since it is essential to perform anextraction process immediately before the injection into the HPLC, it issignificantly difficult to treat a large amount of specimencollectively. Herein, each temperature indicates a storage temperatureof the hexane extraction liquid in FIG. 3.

DISCLOSURE OF THE INVENTION

It is a general object of the present invention to provide an analysismethod and analysis system for coenzyme Q-10 and a 2-electron reducedform thereof which can solve the problems of the conventional techniquesas described above.

A more specific object of the present invention is to realize ananalysis method and analysis system which can accurately quantify thecontents of coenzyme Q-10 and a 2-electron reduced form thereof in aspecimen.

For achieving the objects, as a result of keen examinations by theapplicants, it has been found that the employment of isopropyl alcoholas an extraction solvent used for the pretreatment instead ofmethanol/hexane is preferable. The following invention is made based onthis evidence.

An analysis method for coenzyme Q-10 and a 2-electron reduced formthereof according to the present invention is characterized in that aspecimen containing at least one of coenzyme Q-10 and a 2-electronreduced form thereof is extracted with an water-soluble organic solventas a pretreatment and the extracted liquid as an analytical sample isanalyzed.

Herein, the coenzyme Q-10 indicates a ubiquinone and the 2-electronreduced form of coenzyme Q-10 indicates a ubiquinol, as described above.The water-soluble organic solvent is preferably isopropyl alcohol but isnot limited to it, and a solvent having a polarity comparable to that ofisopropyl alcohol can be employed. For example, a mixed solvent of whichthe polarity is adjusted by mixing methanol, ethanol, butanol, andn-propyl alcohol, etc., can be employed. Additionally, an analysismethod for an analytical sample after the pretreatment is not limited tothe analysis method according to the present invention as describedbelow, and an appropriate method such as the above-mentionedconventional analysis method can be also employed.

Due to the above-mentioned configuration of the present invention, thevariation in a component until the analysis of an analytical sample canbe suppressed for an accurate analysis. Also, it is not necessary toperform the analysis immediately after extracting a specimen.

In this case, if the extracted liquid is stored at a temperature withina range of the melting point of the extracted liquid through roomtemperature, more preferably a temperature of around 4° C., until theextracted liquid is analyzed, then the variation in a component can besuppressed more certainly, which is preferable. Herein, the meltingpoint of the extracted liquid is substantially equal to the meltingpoint of the water-soluble organic solvent used for the extraction.

Also, in this case, if a preparatory treatment for condensing theanalytical sample (extracted liquid) is performed according to a columnswitching method, then even if the concentrations of coenzyme Q-10 and a2-electron reduced form thereof are low, an accurate and highlysensitive analysis can be performed, which is preferable.

Also, in this case, if an extracted liquid from a specimen containingboth coenzyme Q-10 and a 2-electron reduced form thereof is theanalytical sample and the coenzyme Q-10 and the 2-electron reduced formthereof are separated by a column, further subjected to a reductiontreatment, and subsequently detected by a detector, then it is notnecessary to twice inject the analytical sample into a HPLC as in theconventional technique and an efficient analysis can be performed, whichis preferable.

Also, in order to preferably realize the analysis method according tothe present invention, an analysis system for coenzyme Q-10 and a2-electron reduced form thereof is an analysis system used for theanalysis method for coenzyme Q-10 and a 2-electron reduced form thereof,characterized by having a liquid-sending mechanism that includes a firstseries for liquid-sending an analytical sample with a first mobile phaseand a second series for liquid-sending only a second mobile phase, aswitching mechanism for switching liquid-sending routes for the mobilephases of the two series of the liquid-sending mechanisms, acondensation column for receiving the second mobile phase after themobile phase of the first series is received so as to condense theanalytical sample, a separation column for receiving and separatingliquid sent from the condensation column, a reduction column forreceiving and reducing liquid sent from the separation column, and anelectrochemical detector for detection-processing liquid sent from thereduction column.

In this case, if further having an ultraviolet absorption detector as adetector, a simultaneous analysis of a component in a specimen whichcomponent cannot be detected with high sensitivity by electrochemicaldetection, such as cholesterol, etc., can be performed, which ispreferable.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects, features and advantages of the present invention willbecome more apparent when reading the following detailed descriptionwith reference to the accompanying drawings, in which:

FIG. 1 is a diagram showing a general configuration of a conventionalanalysis system for coenzyme Q-10 and a 2-electron reduced form thereof;

FIG. 2 is one example of a chromatogram obtained by an analysis systemin FIG. 1;

FIG. 3A is a graph diagram showing variation in a component with timewhen a specimen under test for the analysis system in FIG. 1 is stored,with respect to a 2-electron reduced form of coenzyme Q-10;

FIG. 3B is a graph diagram showing variation in a component with timewhen a specimen under test for the analysis system in FIG. 1 is stored,with respect to coenzyme Q-10;

FIG. 4 is a diagram for illustrating a pretreatment process in ananalysis method for coenzyme Q-10 and a 2-electron reduced form thereof;

FIG. 5A is a graph diagram showing variation in a component with timewhen an extracted liquid is stored after a pretreatment in an analysismethod according to the present example, with respect to the quantitiesof coenzyme Q-10 and a 2-electron reduced form thereof;

FIG. 5B is a graph diagram showing variation in a component with timewhen an extracted liquid is stored after a pretreatment in an analysismethod according to the present example, with respect to the molarfraction of coenzyme Q-10;

FIG. 6 is a diagram showing a general configuration of an analysissystem for coenzyme Q-10 and a 2-electron reduced form thereof;

FIG. 7 is one example of a chromatogram obtained by the analysis systemin FIG. 6, when blood plasma is employed as a specimen; and

FIG. 8 is one example of a chromatogram obtained by the analysis systemin FIG. 6, when saliva is employed as a specimen.

BEST MODE FOR CARRYING OUT THE INVENTION

Next, a preferred embodiment (referred to as an example, below) of ananalysis method and analysis system for coenzyme Q-10 and a 2-electronreduced form thereof according to the present invention is describedwith reference to the drawings.

In the first example described below, human blood plasma is employed asa specimen containing coenzyme Q-10 and a 2-electron reduced formthereof. Also, in the second example, human saliva is employed as aspecimen containing coenzyme Q-10 and a 2-electron reduced form thereof.

First, a pretreatment in the analysis method for coenzyme Q-10 and a2-electron reduced form thereof according to the first example isdescribed with reference to FIG. 4.

50 μl of human blood plasma as a specimen is sampled (S-1 in FIG. 4),950 μl of isopropyl alcohol is added into it (S-2 in FIG. 4), and thenthey are mixed sufficiently. Subsequently, they are subjected to acentrifugal separator at a rotational speed of 12,000 rpm for 3 minutesand at a temperature of 4° C. Thus, coenzyme Q-10 and a 2-electronreduced form thereof are extracted from the specimen into an isopropylalcohol phase (S-3 in FIG. 4).

When an extracted liquid into which the coenzyme Q-10, etc., has beenextracted is stored at a temperature around 4° C. (S-4 in FIG. 4), the2-electron reduced form of coenzyme Q-10 is rarely oxidized even afterat least 11 hours, as shown in FIG. 5. Thus, the alteration of a storedanalytical sample can be prevented. Additionally, the longitudinal axisin regard to coenzyme Q-10 in FIG. 5(b) represents the ratio(molar-ratio) of the coenzyme Q-10 occupied in the total quantity of thecoenzyme Q-10 and the 2-electron reduced form thereof.

Due to the pretreatment method of the first example according to thepresent embodiment as described above, an analytical sample obtained byextracting coenzyme Q-10 and a 2-electron reduced form thereof from aspecimen can be stored at a stable state for a long period of time.Then, as the extracted liquid is an analytical sample, an accurateanalysis can be provided. Accordingly, for example, a large amount ofspecimen can be continuously and automatically processed by setting theextracted liquid as an analytical sample to an auto-sampler of which thetemperature is controlled at 4° C.

Herein, the temperature of 4° C. is a usual operating temperature when abiological sample is the specimen, and the alteration of othercomponents can be prevented by keeping the biological sample at thistemperature. If the biological sample is at a temperature significantlyover this temperature, the alteration of a component such as the2-electron reduced form of coenzyme Q-10 cannot be avoided. On the otherhand, if the biological sample is frozen and stored, a step ofdefrosting the biological sample is required for analysis and thecontinuous and automatic process using the above-mentioned auto-sampler,etc., cannot be easily realized.

Therefore, the temperature of around 4° C. in the present invention isintended to include both a temperature higher than 4° C. and atemperature lower than 4° C., as long as the biological sample asdescribed above can be maintained without alteration. That is, thestorage temperature of the extracted liquid may be within a range of themelting point of a water-soluble organic solvent for extraction throughordinary temperature. In the first example, since isopropyl alcohol isemployed as the water-soluble organic solvent for extraction, the lowerlimit of the storage temperature results in −89.5° C. that is themelting point of isopropyl alcohol.

Next, a method for analyzing an extracted liquid stored for a requiredtime as an analytical sample by HPLC is described below.

First, an analysis system used for an analysis method according to thepresent invention is described with reference to FIG. 6. An analysissystem 10 according to the present embodiment includes a liquid-sendingmechanism 12, a switching mechanism 14, a concentration column 16, aseparation column 18, a reduction column 20, an electrochemical detector22, and an ultraviolet absorption detector 24.

The liquid-sending mechanism 12 is composed of two series, i.e., thefirst and second series, and each series includes a storage container 25a, 25 b for a mobile phase, and a pump 26 a, 26 for liquid-sending themobile phase of the storage container 25 a, 25 b. The first seriesfurther includes a sample injector 28.

In the storage container 25 a of the first series, a mixed solution ofmethanol and water (an aqueous solution of 95% ethanol), which contains50 mM of sodium perchlorate, is stored as the mobile phase. On the otherhand, in the storage container 25 b of the second series, a mixedsolution of methanol and isopropyl alcohol (a solution of 90% ethanol),which contains 50 mM of sodium perchlorate, is stored as the mobilephase.

For either of the pumps 26 a, 26 b, for example, Inert Pump 3001produced by Shiseido Co., Ltd. can be employed. This Inert Pump 3001 isa constant flow rate and constant pressure type dual piston pump due toa pulse motor, in which the flux rate is 1 through 3,000 μl/min and theupper limit of the discharge pressure is 35 MPa.

The sample injector 28 provided in the first series is for entraining ananalytical sample with the mobile phase of the first series, for whichan appropriate apparatus can be employed and an auto-sampler ispreferably employed.

Where an auto-sampler is employed as the sample injector 28, forexample, Autosampler 3023 produced by Shiseido Co., Ltd. can beemployed. In regard to the Autosampler 3023, the sample injectionquantity is 0.1 through 400 μl (controllable by 0.1 μl), the number oftreated samples is 100 through 200, and the temperature can becontrolled within a range of 4 through 20° C. by means of electroncooling.

The switching mechanism 14 is for switching liquid-sending routes forthe mobile phases of the two series of the liquid-sending mechanism 12.In a mode indicated by an arrow A1 (referred to as mode Al, below) inFIG. 6, the mobile phase of the first series is sent to theconcentration column 16 and liquid exiting from the concentration column16 in which coenzyme Q-10, etc., is eliminated is ejected. On the otherhand, the mobile phase of the second series bypasses the column 16 andis directly sent to the separation column 18.

On the contrary, in a mode indicated by an arrow A2 (referred to as modeA2, below), the mobile phase of the first series is ejected out of thesystem so as to stop the liquid-sending to the concentration column 16,and the mobile phase of the second series is sent to the concentrationcolumn 16, whereby liquid entraining a concentrated analytical sample(liquid based on the second mobile phase) is sent from the concentrationcolumn 16 to the separation column 18. Thus, the concentrated analyticalsample is entrained with the mobile phase of the second series and sentto the separation column 18.

The switching mechanism 14 is composed of, for example, a switchingvalve and, as such a switching valve, for example, a High-PressureSix-Way Switching Valve 3011 produced by Shiseido Co., Ltd. can beemployed. The High-Pressure Six-Way Switching Valve 3011 is a SUS 6-port2-position valve and the withstand pressure thereof is 35 MPa.Additionally, the connection relation of pipes and the like used in theanalysis system 10, which include pipes and the like used in thatswitching mechanism 14, is apparent from FIG. 6 and SUS is employed as amaterial for the pipes in order to prevent the adsorption of thecoenzyme Q-10 and the 2-electron reduced form thereof.

The concentration column 16 is for concentrating the analytical sampleusing, for example, the adsorption effect of filler, etc., and, forexample, LC-8 produced by SUPELCO can be employed, as previouslydescribed.

The separation column 18 is for separating liquid sent out from theconcentration column 16 and, for example, LC-8 produced by SUPELCO canbe employed.

The reduction column 20 is for reducing liquid sent out from theseparation column and, specifically, a column intended to covert thecoenzyme Q-10 in the analytical sample to the 2-electron reduced formthereof. As the reduction column 20, for example, SHISEIDO CQ producedby Shiseido Co., Ltd. can be employed.

The electrochemical detector 22 is intended to analyze the 2-electronreduced form of coenzyme Q-10, which can be detected electrochemicallyand with high sensitivity, and, for example, Electrochemical Detector3005 produced by Shiseido Co., Ltd. can be employed. The ElectrochemicalDetector 3005 is a three-pole potentiostatic type and an applied voltagecan be set digitally by 10 mV within a range of ±1990 mV.

The ultraviolet absorption detector 24 is for analyzing a component witha high absorption'sensitivity to ultraviolet rays such as cholesterolcontained in blood plasma being the analytical sample according to need.As the ultraviolet absorption detector 22, for example, an UV-VISDetector 3002 produced by Shiseido Co., Ltd. can be employed. The UV-VISDetector 3002 is a double-beam single-cell type and the wavelength rangeis 195 through 700 nm.

40 μl of the above-mentioned extracted liquid after the pretreatment isprepared as the analytical sample. 2.0 μl of human blood plasma iscontained in the 40 μl analytical sample.

First, the switching mechanism 14 is set to the mode A2 and the mobilephase of the second series (a solution of 90% methanol) stored in thestorage container 25 b is flown through respective columns of theconcentration column 16, the separation column 18, and the reductioncolumn 20 using the pump 26 b, so that the respective columns arestabilized. Then, liquid-sending to the separation column 18 and thereduction column 20 may be made at mode A1 according to need.

Basically, this column stabilizing operation has only to be performedonce and, subsequently, even if plural analytical samples are analyzedsequentially, it is not necessary to repeat the above-mentionedstabilizing operation each time. However, in the column switchingmethod, the mobile phase of the second series may be sent to each columnbefore and after switching of the switching mechanism 14, for thepurpose of performing an analysis at a more stable state.

Subsequently, the mobile phase of the first series (an aqueous solutionof 95% methanol) stored in the storage container 25 a is liquid-sent tothe concentration column 16 using the pump 26 a, which entrains theabove-mentioned analytical sample using the sample injector 28 inmid-flow. Then, the flow rate is 400 μl/min and the discharge pressureof the pump is 1 MPa. In the concentration column 16, main componentssuch as coenzyme Q-10 and a 2-electron reduced form thereof in theanalytical sample are retained. The residual liquid is ejected out ofthe system.

Subsequently, the mobile phase of the second series (the solution of 90%methanol) stored in the storage container 25 b is liquid-sent to theconcentration column 16 in which the above-mentioned main components areretained, using the pump 26 b. Then, the flow rate is 800 μl/min and thedischarge pressure of the pump is 10.1 MPa.

Additionally, these operations are performed using the switchingmechanism 14 as described above.

Herein, when an auto-sampler is employed as the sample injector 28 so asto process the analytical sample kept at a temperature of 4° C.continuously and automatically, it is more preferable to liquid-send themobile phase of the second series to a column beyond the separationcolumn 18 constantly through the switching mechanism 14 while theconcentrated analytical sample is not sent. Then, the flow rate is 800μl/min and the discharge pressure of the pump is 7.6 MPa.

Thus, the liquid entraining the analytical sample in which the maincomponents are concentrated is liquid-sent to the separation column 18and the reduction column 20 in series, the separation of the maincomponents (as object components for analysis) based on the coenzymeQ-10 and the 2-electron reduced form thereof in the analytical sample isperformed, and further a component to be reduced based on the coenzymeQ-10 is reduced.

The liquid treated in the reduction column 20 is sent to theelectrochemical detector 22 and the ultraviolet absorption detector 24in series and detection-processed.

One example of a chromatogram using human blood plasma as a specimen,which is obtained by the electrochemical detector 22, is shown in FIG.7. The set applied voltage of the electrochemical detector 22 is 600 mV.In FIG. 7, arrow 1 a denotes a peak of the 2-electron reduced form ofcoenzyme Q-10 (ubiquinol) and arrow 2 a denotes a peak of the coenzymeQ-10 (ubiquinone).

Next, the pretreatment in the analysis method for coenzyme Q-10 and a2-electron reduced form thereof according to the second example isdescribed. Since only the specimen is different from that of theabove-mentioned first example (the human blood plasma in the firstexample), the description is provided using FIG. 4 similar to thedescription for the first example.

40 μl of human saliva as a specimen is sampled (S-1 in FIG. 4), a 950 μlof isopropyl alcohol is added into it (S-2 in FIG. 4), and then they aremixed sufficiently. Subsequently, they are subjected to a centrifugalseparator at a rotational speed of 12,000 rpm for 3 minutes and at atemperature of 4° C. Thus, coenzyme Q-10 and a 2-electron reduced formthereof are extracted from the specimen to an isopropyl alcohol phase(S-3 in FIG. 4).

Then, an extracted liquid in which the coenzyme Q-10, etc., has beenextracted is stored at a temperature around 4° C. (S-4 in FIG. 4).Similar to the first example, the alteration of an analytical sampleduring storage can also be prevented in the second example. Therefore,the analytical sample in which coenzyme Q-10 and a 2-electron reducedform thereof have been extracted can be also stored at a stable statefor a long period of time in the second example. Then, the extractedliquid is the analytical sample used to perform an accurate analysis.Additionally, since other conditions are identical to those of the firstexample as previously described, the description of them is omitted.

Next, a method for analyzing the extracted liquid subjected to theabove-mentioned pretreatment and stored for a required time as ananalytical sample using a HPLC is described below.

An analysis method in the second example of the present embodiment isbasically identical to the analysis method in the first exampledescribed above. Therefore, the analysis method in the second example issimply described. Additionally, since those described previously withreference to FIG. 6 are used as a HPLC and analysis system used foranalysis without change, the description for the HPLC and analysissystem is omitted.

40 μl of the extracted liquid after the above-mentioned pretreatment isprepared as an analytical sample. The 40 μl of the analytical samplecontains 2.0 μl human saliva.

First, the switching mechanism 14 is set to the mode A2 and the mobilephase of the second series (a solution of 90% methanol) stored in thestorage container 25 b is flown through respective columns of theconcentration column 16, the separation column 18, and the reductioncolumn 20 using the pump 26 b, so that the respective columns arestabilized. Herein, Capcel Pack C18 AQ S5 (commercial name) with adiameter of 2.0 mm×a length of 35 mm can be employed as theconcentration column 16. Also, Capcel Pack C18 AQ S5 (commercial name)with a diameter of 2.0 mm×a length of 250 mm can be employed as theseparation column 18. Further, SHISEIDO CQ (commercial name) with adiameter of 2.0 mm×a length of 20 mm can be employed as the reductioncolumn 20.

Subsequently, the mobile phase of the first series (an aqueous solutionof 95% methanol) stored in the storage container 25 a is liquid-sent tothe concentration column 16 using the pump 26 a, which entrains theabove-mentioned analytical sample using the sample injector 28 inmid-flow. Then, the flow rate is, for example, 200 μl/min. In theconcentration column 16, main components such as coenzyme Q-10 and a2-electron reduced form thereof in the analytical sample are retained.The residual liquid is ejected out of the system.

Subsequently, the mobile phase of the second series (the solution of 90%methanol) stored in the storage container 25 b is liquid-sent to theconcentration column 16 in which the above-mentioned main components areretained, using the pump 26 b.

Herein, an auto-sampler is employed as the sample injector 28 and theanalytical sample kept at a temperature of 4° C. is continuouslyprovided to the separation column 18. Thus, the liquid entraining theanalytical sample in which the main components are concentrated isliquid-sent to the separation column 18 and the reduction column 20 inseries, the separation of the main components (as object components foranalysis) based on the coenzyme Q-10 and the 2-electron reduced formthereof in the analytical sample is performed, and further a componentto be reduced based on the coenzyme Q-10 is reduced. Then, the flow rateis, for example, 400 μl/min. The liquid treated in the reduction column20 is sent to the electrochemical detector 22 and the ultravioletabsorption detector 24 in series and detection-processed.

One example of a chromatogram using human saliva as a specimen, which isobtained by the electrochemical detector 22, is shown in FIG. 8. In FIG.8, arrow 3 a denotes a peak of the coenzyme Q-10.

According to the analytical method and analytical system for coenzymeQ-10 and a 2-electron reduced form thereof in the first and secondexamples as described above, since extracted liquid in which coenzymeQ-10 and 2-electron reduced form thereof have been extracted from aspecimen is an analytical sample and a concentrated analytical sample isanalyzed, an accurate and highly sensitive analysis can be performed.

Also, according to the analytical method and analytical system forcoenzyme Q-10 and a 2-electron reduced form thereof in the first andsecond examples, coenzyme Q-10 and a 2-electron reduced form thereof areanalyzed simultaneously and, preferably, since a large number ofspecimens are treated continuously and automatically, an efficientanalysis can be performed.

Further, in the analytical method and analytical system for coenzymeQ-10 and a 2-electron reduced form thereof in the second example, salivais employed as a specimen. Since the sampling of saliva is noninvasivein contrast to the sampling of blood (plasma), it is possible to performself-sampling and the amounts of coenzyme Q-10 and a 2-electron reducedform thereof inside the body can be easily detected.

As described above, according to an analytical method for coenzyme Q-10and a 2-electron reduced form thereof of the present invention, since aspecimen containing at least one of coenzyme Q-10 and a 2-electronreduced form thereof is extracted with a water-soluble organic solventas a pretreatment and the extracted liquid is analyzed as an analyticalsample, an accurate analysis can be performed. Also, it is not necessaryto perform an analysis immediately after sampling the specimen.

Also, according to an analytical method for coenzyme Q-10 and a2-electron reduced form thereof of the present invention, sincepreparatory treatment for concentrating an analytical sample by a columnswitching method is performed, even if the concentrations of coenzymeQ-10 and a 2-electron reduced form thereof in the analytical sample arelow, an analysis can be performed accurately and with high sensitivity.

Also, according to an analytical method for coenzyme Q-10 and a2-electron reduced form thereof of the present invention, sinceextracted liquid from a specimen containing both coenzyme Q-10 and a2-electron reduced form thereof is an analytical sample, and thecoenzyme Q-10 and the 2-electron reduced form thereof are separated,further subjected to a reduction treatment, and subsequently detected bya detector, an efficient analysis can be performed.

Also, according to an analytical system for coenzyme Q-10 and a2-electron reduced form thereof of the present invention, since aliquid-sending mechanism that includes the first series forliquid-sending an analytical sample with the first mobile phase and thesecond series for liquid-sending only the second mobile phase, aswitching mechanism for switching liquid-sending routes for the mobilephases of the two series of the liquid-sending mechanisms, acondensation column for receiving the second mobile phase after themobile phase of the first series is received so as to condense theanalytical sample, a separation column for receiving and separatingliquid sent from the condensation column, a reduction column forreceiving and reducing liquid sent from the separation column, and anelectrochemical detector for detection-processing liquid sent from thereduction column are included, an analysis method of the presentinvention can be preferably realized.

1. An analysis method for coenzyme Q-10 and a 2-electron reduced formthereof which quantifies coenzyme Q-10 and a 2-electron reduced formthereof comprised in a specimen, characterized in that the specimencomprising at least one of the coenzyme Q-10 and the 2-electron reducedform thereof is extracted with a water-soluble organic solvent as apretreatment and extracted liquid as an analytical sample is analyzed.2. An analysis method for coenzyme Q-10 and a 2-electron reduced formthereof which quantifies coenzyme Q-10 and a 2-electron reduced formthereof comprised in a specimen, characterized in that the specimencomprising at least one of the coenzyme Q-10 and the 2-electron reducedform thereof is extracted with a water-soluble organic solventcomprising isopropyl alcohol as a pretreatment and extracted liquid asan analytical sample is analyzed.
 3. An analysis method for coenzymeQ-10 and a 2-electron reduced form thereof which quantifies coenzymeQ-10 and a 2-electron reduced form thereof comprised in a specimenaccording to claim 1, further characterized in that the extracted liquidis stored at a temperature within a range of a melting point of theextracted liquid through room temperature until the extracted liquid isanalyzed.
 4. An analysis method for coenzyme Q-10 and a 2-electronreduced form thereof which quantifies coenzyme Q-10 and a 2-electronreduced form thereof comprised in a specimen according to claim 2,further characterized in that the extracted liquid is stored at atemperature within a range of a melting point of the extracted liquidthrough room temperature until the extracted liquid is analyzed.
 5. Ananalysis method for coenzyme Q-10 and a 2-electron reduced form thereofwhich quantifies coenzyme Q-10 and a 2-electron reduced form thereofcomprised in a specimen according to claim 1, further characterized inthat a preparatory treatment for condensing the analytical sampleaccording to a column switching method is performed.
 6. An analysismethod for coenzyme Q-10 and a 2-electron reduced form thereof whichquantifies coenzyme Q-10 and a 2-electron reduced form thereof comprisedin a specimen according to claim 2, further characterized in that apreparatory treatment for condensing the analytical sample according toa column switching method is performed.
 7. An analysis method forcoenzyme Q-10 and a 2-electron reduced form thereof which quantifiescoenzyme Q-10 and a 2-electron reduced form thereof comprised in aspecimen according to claim 1, further characterized in that theextracted liquid is stored at a temperature within a range of a meltingpoint of the extracted liquid through room temperature until theextracted liquid is analyzed, and a preparatory treatment for condensingthe analytical sample according to a column switching method isperformed.
 8. An analysis method for coenzyme Q-10 and a 2-electronreduced form thereof which quantifies coenzyme Q-10 and a 2-electronreduced form thereof comprised in a specimen according to claim 2,further characterized in that the extracted liquid is stored at atemperature within a range of a melting point of the extracted liquidthrough room temperature until the extracted liquid is analyzed, and apreparatory treatment for condensing the analytical sample according toa column switching method is performed.
 9. An analysis method forcoenzyme Q-10 and a 2-electron reduced form thereof which quantifiescoenzyme Q-10 and a 2-electron reduced form thereof comprised in aspecimen according to claim 1, further characterized in that extractedliquid from a specimen comprising both the coenzyme Q-10 and the2-electron reduced form thereof is the analytical sample, and thecoenzyme Q-10 and the 2-electron reduced form thereof are separated by acolumn, further subjected to reduction treatment, and subsequentlydetected by a detector.
 10. An analysis method for coenzyme Q-10 and a2-electron reduced form thereof which quantifies coenzyme Q-10 and a2-electron reduced form thereof comprised in a specimen according toclaim 2, further characterized in that extracted liquid from a specimencomprising both the coenzyme Q-10 and the 2-electron reduced formthereof is the analytical sample, and the coenzyme Q-10 and the2-electron reduced form thereof are separated by a column, furthersubjected to reduction treatment, and subsequently detected by adetector.
 11. An analysis method for coenzyme Q-10 and a 2-electronreduced form thereof which quantifies coenzyme Q-10 and a 2-electronreduced form thereof comprised in a specimen according to claim 1,further characterized in that the extracted liquid is stored at atemperature within a range of a melting point of the extracted liquidthrough room temperature until the extracted liquid is analyzed,extracted liquid from a specimen comprising both the coenzyme Q-10 andthe 2-electron reduced form thereof is the analytical sample, and thecoenzyme Q-10 and the 2-electron reduced form thereof are separated by acolumn, further subjected to reduction treatment, and subsequentlydetected by a detector.
 12. An analysis method for coenzyme Q-10 and a2-electron reduced form thereof which quantifies coenzyme Q-10 and a2-electron reduced form thereof comprised in a specimen according toclaim 2, further characterized in that the extracted liquid is stored ata temperature within a range of a melting point of the extracted liquidthrough room temperature until the extracted liquid is analyzed,extracted liquid from a specimen comprising both the coenzyme Q-10 andthe 2-electron reduced form thereof is the analytical sample, and thecoenzyme Q-10 and the 2-electron reduced form thereof are separated by acolumn, further subjected to reduction treatment, and subsequentlydetected by a detector.
 13. An analysis method for coenzyme Q-10 and a2-electron reduced form thereof which quantifies coenzyme Q-10 and a2-electron reduced form thereof comprised in a specimen according toclaim 1, further characterized in that a preparatory treatment forcondensing the analytical sample according to a column switching methodis performed, extracted liquid from a specimen comprising both thecoenzyme Q-10 and the 2-electron reduced form thereof is the analyticalsample, and the coenzyme Q-10 and the 2-electron reduced form thereofare separated by a column, further subjected to reduction treatment, andsubsequently detected by a detector.
 14. An analysis method for coenzymeQ-10 and a 2-electron reduced form thereof which quantifies coenzymeQ-10 and a 2-electron reduced form thereof comprised in a specimenaccording to claim 2, further characterized in that a preparatorytreatment for condensing the analytical sample according to a columnswitching method is performed, extracted liquid from a specimencomprising both the coenzyme Q-10 and the 2-electron reduced formthereof is the analytical sample, and the coenzyme Q-10 and the2-electron reduced form thereof are separated by a column, furthersubjected to reduction treatment, and subsequently detected by adetector.
 15. An analysis method for coenzyme Q-10 and a 2-electronreduced form thereof which quantifies coenzyme Q-10 and a 2-electronreduced form thereof comprised in a specimen according to claim 1,further characterized in that the extracted liquid is stored at atemperature within a range of a melting point of the extracted liquidthrough room temperature until the extracted liquid is analyzed, apreparatory treatment for condensing the analytical sample according toa column switching method is performed, extracted liquid from a specimencomprising both the coenzyme Q-10 and the 2-electron reduced formthereof is the analytical sample, and the coenzyme Q-10 and the2-electron reduced form thereof are separated by a column, furthersubjected to reduction treatment, and subsequently detected by adetector.
 16. An analysis method for coenzyme Q-10 and a 2-electronreduced form thereof which quantifies coenzyme Q-10 and a 2-electronreduced form thereof comprised in a specimen according to claim 2 ,further characterized in that the extracted liquid is stored at atemperature within a range of a melting point of the extracted liquidthrough room temperature until the extracted liquid is analyzed, apreparatory treatment for condensing the analytical sample according toa column switching method is performed, extracted liquid from a specimencomprising both the coenzyme Q-10 and the 2-electron reduced formthereof is the analytical sample, and the coenzyme Q-10 and the2-electron reduced form thereof are separated by a column, furthersubjected to reduction treatment, and subsequently detected by adetector.
 17. An analysis system used for analysis of coenzyme Q-10 anda 2-electron reduced form thereof, comprising a liquid-sending mechanismcomprising a first series for liquid-sending an analytical sample with afirst mobile phase and a second series for liquid-sending only a secondmobile phase, a switching mechanism for switching liquid-sending routesfor the mobile phases of the two series of the liquid-sending mechanism,a condensation column for receiving the second mobile phase after themobile phase of the first series is received so as to condense theanalytical sample, a separation column for receiving and separatingliquid sent from the condensation column, a reduction column forreceiving and reducing liquid sent from the separation column, and anelectrochemical detector for detection-processing liquid sent from thereduction column.
 18. An analysis system used for analysis of coenzymeQ-10 and a 2-electron reduced form thereof according to claim 17,further comprising an ultraviolet absorption detector as a detector.